Chromosome anomaly affects over 20% of oocytes obtained from women in their early thirties and this prevalence more than doubles as women enter their forties. These chromosome abnormalities are almost always lethal to the developing embryo and their high prevalence is responsible for many failed in vitro fertilization (IVF) treatments. Consequently the identification of chromosomally normal oocytes is of great importance for IVF treatment.
A number of clinical (e.g. preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS)) and scientific studies, employing various cytogenetic techniques, have demonstrated that in patients with indications for PGD and PGS, at least two-thirds of human preimplantation embryos contain aneuploid cells (Delhanty et al., 1997, Human Genetics 99:755-760; Munné and Cohen, 1998, Human Reproduction Update 4:842-855; Wells and Delhanty, 2000, Mol Hum Repro 6:1055-1062; Voullaire et al., 2002, Mol Hum Repro 8:1035-1041; Voullaire et al., 2000, Hum Gen 106:210-217; Coonen et al., 2001, Hum Repro 19:316-324; Baart et al., 2007, Prenatal Diag 27:55-63). It is has also been demonstrated that 85% of embryos produced in vitro and transferred into the uterus fail to develop into an infant, leaving only a small fraction destined to become a live birth (Kovalevsky and Patrizio, 2005, Fertility and Sterility 84:325-330). To address this low competence rate, usually multiple oocytes and embryos are produced during IVF. But to minimize the risk of a high-order multiple pregnancy (e.g. triplets, quads, etc) usually only 2 or 3 embryos are transferred to the uterus. A great challenge for physicians is identifying which embryos are the most likely to result in a pregnancy and ensure that these embryos are among the limited number selected for transfer to the uterus.
Methods for identifying healthy oocytes and embryos based upon morphological assessments have not been very successful (for review, see Patrizio et al., 2007, Repro BioMedicine Online 15:346-353). In addition to morphological assessments, some clinics also employ cytogenetic assessments. Oocytes can be tested for aneuploidy by biopsy of the first and second polar bodies and subjecting them to cytogenetic analysis. The detection of extra or missing chromosomes in a polar body is indicative of a reciprocal loss or gain of chromosomes in the corresponding oocyte. Embryos derived from chromosomally normal oocytes can be given priority for transfer during assisted reproduction, potentially improving outcome by avoiding transfer of embryos carrying deleterious aneuploidies. But, classical cytogenetic techniques are difficult to apply to polar bodies, due to problems of obtaining high quality chromosome spreads. For this reason, the vast majority of chromosomal tests performed on polar bodies have employed fluorescence in-situ hybridization (FISH). Using FISH, it is possible to assess 5-12 chromosomes in individual polar bodies/oocytes regardless of chromosome morphology (Verlinsky et al., 1998, J Assisted Repro & Gen 15:285-289; Kuliev et al., 2003, Repro Biomed Online 6:54-59; Pujol et al., 2003, Eur J Hum Gen 11:325-326). However, this method examines only less than half of the chromosomes. In addition, the removal of a cell is an invasive procedure that may damage the embryo.
More recently, comparative genomic hybridization (CGH) has been used to assess the copy number of chromosomes in polar bodies and oocytes, although to date most analyses have been performed in a research context (Wells et al., 2002, Fertility and Sterility 78:543-549; Gutierrez-Mateo et al., 2004, Hum Repro 19:2118-2125; Fragouli et al. 2006, Cyto Gen Res 114:30-38; Fragouli et al., 2006, Hum Repro 21:2319-2328). CGH has the major advantage that every chromosome is analyzed, rather than the limited subset assessed using FISH, but it is a time-consuming and labor-intensive method that is difficult to perform within the limited time available for preimplantation testing.
Studies conducting gene expression analysis using reverse transcription followed by real-time polymerase chain reaction (PCR) have found that specific genes display alterations in activity that may be related to oocyte or embryo quality and competence (Wells et al., 2005, Fertility and Sterility 84:343-355; Dode et al., 2006, Mol Repro Devel 73:288-297; Russell et al., 2006, Mol Repro Devel 73:1255-1270), and that morphologically abnormal preimplantation embryos frequently display atypical patterns of gene expression (Wells et al., 2005, Fertility and Sterility 84:343-355). Despite the accuracy and sensitivity of real-time PCR, the method is limited by the restricted number of genes that can be assessed for each oocyte or embryo, generally less than 10 genes.
Reproductive medicine would benefit greatly from a method capable of the noninvasive characterization and identification of those oocytes or embryos most likely to result in successful fertilization and implantation by measuring the level of marker expression associated with oocyte competence and oocyte incompetence. The present invention fulfills this need.